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High-content imaging, immunoblot and immunofluorescence data related to: Caprin-1 binding to the critical stress granule protein G3BP1 is influenced by pH

Schulte, Tim1; Han, Xiao1; Williams, Lucy1; Kedersha, Nancy2; Fleck, Jonas Simon1; Tan, Timothy J. C.1; Dopico, Xaquin Castro1; Olsson, Anders3; Morro, Ainhoa Moliner1; Hanke, Leo1; Nilvebrant, Johan3; Giang, Kim Anh3; Nygren, Per-Åke3; Anderson, Paul2; Achour, Adnane1; McInerney, Gerald M.1; Panas, Marc1

Published Sep 22, 2023 on Dryad. https://doi.org/10.5061/dryad.k98sf7mb8

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Abstract

G3BP is the central node within stress-induced protein–RNA interaction networks known as stress granules (SGs). The SG-associated proteins Caprin-1 and USP10 bind mutually exclusively to the NTF2 domain of G3BP1, promoting and inhibiting SG formation, respectively. Herein, we present the crystal structure of G3BP1-NTF2 in complex with a Caprin-1-derived short linear motif (SLiM). Caprin-1 interacts with His-31 and His-62 within a third NTF2-binding site outside those covered by USP10, as confirmed using biochemical and biophysical-binding assays. Nano-differential scanning fluorimetry revealed reduced thermal stability of G3BP1-NTF2 at acidic pH. This destabilization was counterbalanced significantly better by bound USP10 than Caprin-1. The G3BP1/USP10 complex immunoprecipated from human U2OS cells was more resistant to acidic buffer washes than G3BP1/Caprin-1. Acidification of cellular condensates by approximately 0.5 units relative to the cytosol was detected by ratiometric fluorescence analysis of pHluorin2 fused to G3BP1. Cells expressing a Caprin-1/FGDF chimera with higher G3BP1-binding affinity had reduced Caprin-1 levels and slightly reduced condensate sizes. This unexpected finding may suggest that binding of the USP10-derived SLiM to NTF2 reduces the propensity of G3BP1 to enter condensates.